Iron ligand mutants in protein R2 of Escherichia coli ribonucleotide reductase

 Ribonucleotide reductase protein R2 contains a diiron-oxo center with the ability to generate and stabilize a catalytically essential tyrosyl radical. The six protein-derived ligands (four carboxylates and two histidines) of the diiron site were, in separate experiments, mutated to alanines and in two cases also to histidines. We found that removal or exchange of an iron ligand did not in general abolish the formation of a diiron site in the mutant proteins, although all mutant proteins lost the bound metal ions with time upon storage. Iron bound to the mutant proteins was characterized by light absorption, EPR and resonance Raman spectroscopy. In addition, the ability of the mutant proteins to form a tyrosyl free radical and the catalytic competence of the latter were determined by EPR spectroscopy and activity measurements. The diiron sites of mutant proteins D84H and E238A were quite reminiscent of that in wild-type R2. Four of the other mutant proteins (H118A, E204A, E204H, H241A) could form the same number of metal sites as wild-type R2, but with different spectroscopic properties. The mutation E115A affecting the only μ-bridging ligand lowered the amount of bound iron to less than half. An important observation was that D84A, H118A and E204A formed transient tyrosyl radicals, but only the E204A mutant protein was enzymatically active. D84A and H118A affect iron ligands which have been suggested to participate in long-range electron transfer during catalysis. Our observation that these mutant proteins are catalytically inert, despite formation of a tyrosyl radical, underscores the necessity for an intact electron transfer pathway for catalytic activity in ribonucleotide reductase. Received: 31 August 1995 / Accepted: 14 February 1996     

The role of the perch effect on the nucleation process in Mediterranean semi-arid oldfields

Oldfield succession in Mediterranean ecosystems has been studied extensively in mesic conditions. However, this phenomenon is still poorly understood in semi-arid Mediterranean areas, where reduced plant cover, the importance of facilitation processes and the role of abiotic factors make these environments distinct. We first test whether the carob tree (Ceratonia siliqua) generates nucleation patterns in semi-arid oldfields, and to what extent such patterns change with abandonment age. Then we test to what extent nucleation can be explained by the perch effect. And finally, we test whether the nucleated pattern around carob trees is a source of diversity in the oldfields studied. To answer these questions we located oldfields abandoned 25 and 50 years ago (20 in each case) in the Alacant Province (SE Spain, Iberian Peninsula) on the basis of aerial photographs and personal interviews with local landowners and managers. In each oldfield woody plant density and richness were sampled on two microsites: under the carob tree and in the open field. Analysis was performed on all woody plants and by separating the species in two functional groups: fleshy-fruited (with fleshy mesocarp) and non-fleshy-fruited species. The results suggest that woody vegetation colonising abandoned C. siliqua fields in SE Spain is not randomly distributed but follows a nucleation pattern with higher plant density under the trees. However, the nucleation pattern is only significant for fleshy-fruited species, suggesting that facilitative interactions alone cannot explain the nucleation pattern and that the perch effect plays an important role. The results also show that the nucleation pattern (total plant density and density of non-fleshy-fruited plants) did not increase with abandonment age, while the perch effect (density of fleshy-fruited plants) did increase significantly. Furthermore, the results also show that the nucleation pattern is not only a loci of high plant density but also a loci of high species richness. Thus we can conclude that the nucleation pattern found in oldfield succession is best explained by the perch effect, while facilitation has a secondary importance. This emphasises the key role that dispersal mode has on the dynamics of vegetation recovery in formerly cropped areas.     

Continued Decline of Malaria in The Gambia with Implications for Elimination

Background

A substantial decline in malaria was reported to have occurred over several years until 2007 in the western part of The Gambia, encouraging consideration of future elimination in this previously highly endemic region. Scale up of interventions has since increased with support from the Global Fund and other donors.

Methodology/Principal Findings

We continued to examine laboratory records at four health facilities previously studied and investigated six additional facilities for a 7 year period, adding data from 243,707 slide examinations, to determine trends throughout the country until the end of 2009. We actively detected infections in a community cohort of 800 children living in rural villages throughout the 2008 malaria season, and assayed serological changes in another rural population between 2006 and 2009. Proportions of malaria positive slides declined significantly at all of the 10 health facilities between 2003 (annual mean across all sites, 38.7%) and 2009 (annual mean, 7.9%). Statistical modelling of trends confirmed significant seasonality and decline over time at each facility. Slide positivity was lowest in 2009 at all sites, except two where lowest levels were observed in 2006. Mapping households of cases presenting at the latter sites in 2007–2009 indicated that these were not restricted to a few residual foci. Only 2.8% (22/800) of a rural cohort of children had a malaria episode in the 2008 season, and there was substantial serological decline between 2006 and 2009 in a separate rural area.

Conclusions

Malaria has continued to decline in The Gambia, as indicated by a downward trend in slide positivity at health facilities, and unprecedented low incidence and seroprevalence in community surveys. We recommend intensification of control interventions for several years to further reduce incidence, prior to considering an elimination programme.     

Metabolism of glucose 1,6-P2. I. Enzymes involved in the synthesis of glucose 1,6-P2 in pig tissues

Pig tissues show four enzymatic activities of glucose 1,6-P2 synthesis: (A) 2 [glucose 1-P]----glucose 1,6-P2 + glucose; (B) glucose 1-P + ATP----glucose 1,6-P2 + ADP; (C) glucose 1-P + fructose 1,6-P2----glucose 1,6-P2 + fructose 6-P; (D) glucose 1-P + glycerate 1,3-P2----glucose 1,6-P2 + glycerate 3-P. Brain is the tissue with highest capability of glucose 1,6-P2 synthesis. With the exception of skeletal muscle, activity "D" represents the highest activity of glucose 1,6-P2 synthesis. In muscle, activity "B" is the major activity. The existence of a specific glucose 1,6-P2 synthase which catalyzes reaction "D" is confirmed. Two peaks of such an enzyme are isolated by ion-exchange chromatography. There is an enzyme which specifically catalyzes reaction "C", not previously described. There is a glucose 1-P kinase not identical to phosphofructokinase.     

Intraspecific variation and plasticity in growth and foliar morphology along a climate gradient in the Canary Island pine

Foliar plasticity in response to ontogeny, location within the plant and environmental changes is widespread among long-lived organisms. To quantify the phenotypic variation in needle morphology and anatomy in response to a climate gradient, we compared contrasted populations of Pinus canariensis grown in five sites inside and outside the natural distribution area of the species. Most needle and growth traits were strongly affected by site. In general, site xericity increased the relative area of the dermal and transfusion tissues and decreased mesophyll and endodermis. Within each site, provenances from less productive locations tended to show longer needles, less relative area of dermal tissues but higher relative area of mesophyll and transfusion tissue than provenances from fertile origins. Although sclerophylly increased with aridity, no genetic differences were found for this trait thus apparently the ontogenetic delay of some provenances in xeric environments was not related with the formation of tougher needles. Several patterns of phenotypic response to different environments were shown. In general, all traits were plastic but the degree of plasticity was higher in traits related with growth than foliar traits. These results, combined with formerly published research, suggest that highly plastic populations rather than narrowly specialized ones have been selected in this species to cope with the complex interaction of environmental factors in its habitat.     

Glucose phosphate isomerase deficiency: enzymatic and familial characterization of Arg346His mutation

Homozygous glucose phosphate isomerase (GPI) deficiency is one of the most important genetic disorders responsible for chronic non-spherocytic hemolytic anemia (CNSHA), a red blood cell autosomal recessive genetic disorder which causes severe metabolic alterations. In this work, we studied a patient with CNSHA due to an 82% loss of GPI activity resulting from the homozygous missense replacement in cDNA position 1040G>A, which leads to substitution of the protein residue A346H mutation. The enzyme is present in a dimeric form necessary for normal activity; the A346H mutation causes a loss of GPI capability to dimerize, which renders the enzyme more susceptible to thermolability and produces significant changes in erythrocyte metabolism.     

Shoot growth components and flowering phenology in grafted<Emphasis Type="Italic"> Pinus halepensis</Emphasis> Mill.

Shoot elongation and flowering were assessed for a season (January–November) in 25 grafts from five clones of Pinus halepensis growing in a seed orchard. A co-dominant shoot from the upper crown and a dominated, low shoot were measured from each ramet. Upper shoots elongated continuously from a variable onset date between January and March and followed a logistic function against Julian day and a Gompertz function against heat sum above 0°C. Three to seven (averaging five) successive cycles were formed through the growing season; usually, two of them were preformed in the terminal bud (spring cycles) and one to four were neo-formed, summer cycles. The number of summer cycles and their contribution to the annual shoot growth were the only variables with a significant clonal influence. Ovulate strobili appeared from February to April and in October. Some ramets showing two female flowering cycles in the same shoot were observed. Lower shoots, bearing pollinate strobili always displayed a single spring cycle preformed in the winter bud.     

Fetal anasarca (Hydrops foetalis) associated with lymphoid tissue agenesis possibly due to an autosomal recessive gene defect in sheep

Fourteen hydrops fetalis cases appeared in a sheep flock in the Soria province of Central Spain in two lambing seasons in 2000. There were no previous cases of hydrops fetalis in this flock. Normal delivery could not be completed because fetal weights ranged from 12 to 16 kg and fetuses had massive subcutaneous edema. Five affected pregnant females were studied. The complete lack of lymph nodes in the fetuses was the most outstanding finding, this anomaly likely being the origin of generalized fluid accumulation. Karyotypes were normal. A blind protocol of parentage testing was performed by means of DNA microsatellite analysis, and one of the five existing rams was found to be the only compatible sire of the affected fetuses. This male had been selected from the same flock while the other rams had all been acquired from other farms. The first cases appeared when this ram began breeding, and no cases were observed after replacing it. Male and female fetuses were affected in similar proportions. The existence of a recessive allele affecting normal lymph node embryonic development in this flock is proposed as the most appropriate hypothesis. As a consequence, the use of rams from different farms is indicated as an efficient emergency measure in similar situations, while the affected flock should be excluded from selection programs as long as the anomalous gene remains unidentified.     

Effects of thyroid hormone and hypoxia on 2,3-bisphosphoglycerate, bisphosphoglycerate synthase and phosphoglycerate mutase in rabbit erythroblasts and reticulocytes in vivo

OBJECTIVES: The effects of triiodothyronine (T(3)) and hypoxia on 2,3-bisphosphoglycerate (2,3-BPG) studied in vitro are unclear. To clarify these effects we selected a more physiologic approach: the in vivo study in rabbits. We also present the changes produced by T(3) and hypoxia on phosphoglycerate mutase (PGAM), which requires 2,3-BPG as a cofactor, and 2,3-BPG synthase (BPGS), the enzyme responsible for 2,3-BPG synthesis in erythroblasts and reticulocytes. METHODS: Hyperthyroidism was induced by daily T(3) injection (250 microg/kg), hypoxia by a mixture of 90% nitrogen and 10% oxygen and hypothyroidism by propylthiouracil (PTU) added to drinking water. RESULTS: Both T(3) administration and hypoxic conditions increased 2,3-BPG levels and BPGS mRNA levels and activity in erythroblasts but not in reticulocytes. Unlike BPGS, both PGAM mRNA levels and activity were increased in erythroblasts and reticulocytes under hyperthyrodism and hypoxia. The antihormone PTU produced opposite effects to T(3). CONCLUSION: The results presented here suggest that both hyperthyroidism and hypoxia modulate in vivo red cell 2,3-BPG content by changes in the expression of BPGS. Similarly, the changes in PGAM activity are also explained by changes in its expression.